The objective of this proposal is to elucidate the mechanisms by which hormones (steroid hormones, thyroidal hormones, and biogenic amines) modulate the intracellular processing, turnover, storage and release of TRH and LHRH from hypothalamic neurons, using synaptosomes and isolated secretory granules as model systems. In order to achieve this goal, we propose (1) to investigate whether hypothalamic granules containing LHRH also contain proLHRH and he enzyme(s) for converting proLHRH to LHRH, and whether hypothalamic granules containing TRH also contain proTRH and enzyme(s) for coverting proTRH to TRH; (2) to define the location within the LHRH granules of the coverting enzyme(s) and proLHRH and the location within the TRH granules of converting enzyme(s) and proTRH; (3) to determine the turnover of LHRH and of TRH in their secretory granules after a prolonged continuous infusion of (3H)amino acids into a ventricle of the brain; (4) to evaluate the role of actomyosin, tubulin, and divalent cations in the release of LHRH and of TRH from isolated secretory granules and from isolated synaptosomes; (5) to analyze whether newly synthesized LHRH or TRH is preferentially released from hypothalamic secretory granules; (6) to investigate whether phosphorylation (catalyzed by cAMP-dependent protein kinase) of granule proteins and of microtubule proteins is an intermediate step in the process of release of LHRH and of TRH from hypothalamic secretory granules. Once these molecular parameters are defined, we will utilize this knowledge to elucidate the mechanisms of hormonal modulation of these intracellular events.